C-terminal α-amidating enzyme (peptidyl-glycine alpha-amidating monooxygenase I, EC 1.14.17.3) is present in eukaryotic organisms and forms the C-terminal amide structure of some biologically active peptides (peptide hormones, neuropeptides, peptide toxins etc.) or proteins. The C-terminal amide structure is known to be indispensable for the expression of biological activities of these peptides or proteins. In the case of human calcitonin, for example, it is known when the native C-terminal proline amide residue is converted to a proline residue, the biological activity decreases to as low as 1/1600 of the original activity.
Also, the Xenopus laevis C-terminal α-amidating enzyme per se has been disclosed in Japanese Patent No. 2598050 (registered on Jan. 9, 1997) and its coding gene has been disclosed in Japanese Patent No. 2581527 (registered on Nov. 21, 1996), respectively.
From the structural analysis of precursors of peptides and proteins having the C-terminal amide structure, it was found that in substrates for C-terminal α-amidating enzymes there is always glycine (Gly) present at the C-terminal end of the residue to be amidated (conversion of a —COOH group to a —CONH2 group), which is represented by a general formula R-X-Gly wherein X represents any amino acid residue to be α-amidated at the C-terminus, Gly represents a glycine residue, and R represents the rest of said peptide or protein. On this Gly, a two-stage reaction of oxidation via a copper ion (first stage: hydroxylation of the α-carbon of Gly) and dealkylation (second stage: release of glyoxylic acid) takes place so that the C-terminus of the substrate is amidated. It is reported that in order to obtain the maximum enzyme activity of this amidating enzyme, ascorbic acid in addition to molecular oxygen and copper ion (Cu2+) are required (see Betty A. Eipper, Richard E. Mains, and Christopher C. Glembotski, “Identification in Pituitary Tissue of a Peptide-amidation Activity That Acts on Glycine-Extended Peptides and Requires Molecular Oxygen, Copper and Ascorbic Acid” Proc. Natl. Acad. Sci. U.S.A. 80, 5144-5148, 1983).
Generally since such modifications including amidation, phosphorylation and acylation take place after translation from mRNA, they are called post-translational modifications, phenomena that are only observed in eucaryotic cells. Prokaryotic cells such as E. coli that is widely used in the production of recombinant proteins and peptides are incapable of such a post-translational modification. Considering the biosynthetic mechanisms of amidated peptides by eucaryotic cells that have been elucidated to date, amidated peptides can be produced in large quantities by gene recombinant technology using prokaryotic cells such as E. coli. 
An amidated peptide can be produced in large quantities and at low cost by a method in which an amidated peptide precursor represented by a general formula R-X-Gly is expressed in large quantities as a recombinant in prokaryotic cells such as E. coli, a C-terminal α-amidating enzyme derived from eucaryotic cells is secured in large quantities, and said amidated peptide precursor is treated with said C-terminal α-amidating enzyme in vitro in an optimal reaction condition for producing an amidated peptide to produce the amidated peptide. In fact, efforts to produce amidated peptides by such a method has been made up to now, as described below.
Unigene Laboratories, Inc., Fairfield, N.J. 07004, “Production of recombinant salmon calcitonin by in vitro amidation of an Escherichia coli produced precursor peptide.” Biotechnology (NY), 1993 January; 11(1):64-70 reports a method in which a salmon calcitonin (sCT) recombinantly produced using Escherichia coli was fused to part of glutathione S-transferase and expressed, sulfonated, and cleaved with cyanogen bromide, and using a C-terminal α-amidating enzyme expressed separately in CHO cells, the C-terminus of sCT was amidated in vitro.
Kokai (Japanese Unexamined Patent Publication) No. 7-163340 also describes a method of producing a human-derived calcitonin (hCT) using an amidating enzyme that was similarly expressed in CHO cells.
In these methods, the C-terminal α-amidating enzymes used in amidating the C-terminus of a protein of interest were produced by the CHO cell which is an animal cell.
Generally, however, the production of a recombinant protein using an animal cell takes a long culturing time and thus poses problems such as low productivity per unit time. As a method for resolving this problem, a method of using E. coli that enables production in a shorter culturing time has been developed as exemplified in Kokai (Japanese Unexamined Patent Publication) No. 7-250691.
This method permits the expression of a Xenopus laevis C-terminal α-amidating enzyme (peptidyl-glycine alpha-amidating monooxygenase I, EC 1.14.17.3) in large quantities by a recombinant technology in E. coli. However, most of the C-terminal α-amidating enzyme and derivatives thereof expressed by this method are forming inclusion bodies (a mass of inactive protein having the same amino acid sequence but does not have a higher-order structure, and thus is called insoluble granules) in E. coli and do not exhibit the activity of the C-terminal α-amidating enzyme.
Thus, an inert enzyme produced by such a method must be converted by some means (for example, refolding) to an active form. For this purpose, in the invention described in Kokai (Japanese Unexamined Patent Publication) No. 7-250691, the C-terminal α-amidating enzyme expressed in E. coli was treated with a denaturing agent such as urea or guanidine hydrochloride, and then was refolded by lowering the concentration of the denaturing agent. However, the activity of the enzyme obtained by this method was about 10-15 mU per mL of the culture liquid, which was lower than that (2,860 U/mL culture liquid) of the amidating enzyme expressed in CHO cells described in the invention of Kokai (Japanese Unexamined Patent Publication) No. 7-163340.